Infection with hepatitis B virus (HBV) is a world-wide public health problem, with chronic carriers accounting for approximately 10 percent of the population of Asia and Africa. Major causes of HBV-associated mortality are: chronic active hepatitis, liver cirrhosis and hepatocellular carcinoma. Both chronic carriers and newly infected individuals are at risk of succumbing to such complications. One important transmission route is the infection of newborn infants at parturition by mothers who have active infections or are chronic carriers. Other routes for transmission include contaminated blood or blood products used to treat other health problems.
On infection with HBV, large quantities of the virus and associated particles are present in the serum. These particles may contain DNA, but are largely empty viral envelopes which have hepatitis B surface antigen (HBsAg) on their surface. The appearance of antibodies to the HBsAg is usually not observed until approximately two months following the disappearance of circulating HBsAg. During this period, a person is highly infectious, but may be clinically diagnosed as non-infectious due to undetectable levels of HBsAg or antibodies to HBsAg. The viral particles present in the serum are known to shed their surface coat exposing the nucleocapsid. Both IgM and IgG class antibodies are produced to a protein of the nucleocapsid core. This protein is known as the hepatitis B core antigen (HBcAg). Early in infection, IgM anti-HBcAg antibodies are produced and their titres rise as circulating HBsAg titers fall. The titer of IgG anti-HBcAg antibodies increases as the titers of the IgM class antibodies fall and before the anti-HBsAg antibody titers rise significantly. It is therefore advantageous to test for the presence of IgM and IgG class antibodies to HBcAg in diagnosing HBV immune status.
Immunoassays have been developed for the detection of IgM anti-HBcAg antibodies and total anti-HBcAg antibodies. The commercially available assays for total anti-HBcAg antibodies are generally competitive assays using labeled human polyclonal antibody to HBcAg collected from infected donors to compete with unlabeled antibodies from the patient sample for binding to a solid phase coated with HBcAg. An example of a commercially available assay of this type is Corzyme.TM. Enzyme Immunoassay (Abbott Laboratories, Chicago, IL). The most common commercially available assays for IgM class antibodies can be called multilayer sandwich assays. A solid phase anti-IgM antibody is used to capture all IgM antibodies from a patient's sample. HBcAg is then added and binds to the specific IgM class anti-HBcAg from the same. Labeled human polyclonal anti-HBcAg is then used to detect any HBcAg bound to the support thus indicating the presence of IgM class anti-HBcAg in the sample. An example of a commercially available assay of this type is Corzyme.TM. M Enzyme Immunoassay (Abbott Laboratories). Both assays use large quantities of Human polyclonal anti-HBcAg. Other formats are possible.
The large amounts of human polyclonal anti-HBcAg antibodies needed for such assays must be collected, preferably from the same donor population each time, isolated, purified and labeled with a radioisotope or enzyme. There is significant health hazard and expense associated with this extensive processing of contaminated blood. It would be advantageous to obtain a constant and consistent supply of antibody to HBcAg without exposure to such a health risk. This need can be met by a monoclonal antibody which can substitute in these immunoassays.
Kohler and Milstein first reported the estabilshment of a continuous hybrid cell line secreting a monoclonal antibody in 1975 [Nature, Volume 256, 495 (1975)]. Since then the process for producing monoclonal antibodies to a variety of antigens has become well known in the art. The production of monoclonal antibodies with specific desirable properties to any given antigen cannot be predicted from the teachings of the art.
Cianfriglia et al. [Hybridoma, Vol. 12(4), 451-457 (1983)] disclose a short-term immunization schedule for producing hybridomas secreting antibodies.
Monoclonal antibodies for HBsAg have been disclosed by many groups (e.g., Wands et al., U.S. Pat. No. 4,271,145 issued June 2, 1981, and U.S. Pat. No. 4,491,632 issued Jan. 1, 1985). Wands et al. report it is critical that the first administration of antigen be given intraperitoneally and second administration be given intravenously at least 3 week later. The antigen used for immunization was isolated from contaminated human plasma. Cell fusion was done by known techniques. Three tests were used to assess anti-HBsAg activity. Binding of antibody to HBsAg coated microtiter plates was probed with either [.sup.125 I]-HBsAg or [.sup.125 I]-goat anti-mouse F(ab').sub.2. The third assay tested the ability of the anti-HBsAg to aggutinate human O-negative red blood cells coated with HBsAg. They suggest that such a process would also be useful for producing monoclonal antibodies to HBcAg.
Tedder et al. [Journal of Hygiene Cambridge, Volume 90, 135-142 (1983) and Proceedings International Hepatitis Workshop, 201-208 (1983)]report the production of six monoclonal antibodies for HBcAg. Mice were immunized with 3 to 4 successive intraperitoneal injections of 50 to 100 .mu.g of HBcAg purified from infected liver. Clones secreting anti-HBcAg were identified using a capture assay comprising: binding all available mouse IgG to a solid phase coated with rabbit anti-mouse IgG, adding purified HBcAg to bind to any anti-HBcAg present, adding [.sup.125 I]-human anti-HBcAg and determining how much radioactivity bound to the support. The presence of radioactivity bound to the support indicated the clone tested was secreting anti-HBcAg. Tedder et al. also state that an alternative assay for detection of anti-HBcAg-secreting hybrids might have been one of the commercially available competitive RIA or ELISA tests. They state however that such an assay would be expected to be of limited value since the monoclonal antibody will block only a single epitope of those present on the solid phase HBcAg. Tedder et al. report testing supernatants from the 6 hybridomas secreting monoclonal anti-HBcAg antibodies using a competitive RIA, but state that the level of inhibition obtained (ranging from 32 to 88%) would not have permitted reliable detection of the antibodies by the competitive RIA alone.
Tedder et al. disclose using the monoclonal anti-HBcAg antibodies in a liver biopsy for presence of HBcAg. They do not disclose use of the antibodies in an inhibition (e.g. competition) assay to detect antibodies to HBcAg in body fluid.
Furuya et al. [Japan J. Med. Sci. Biol., 37, 151-159 (1984)] also report production of monoclonal antibodies to HBcAg. Mice were immunized with HBcAg purified from human plasma by subcutaneous and intramuscular injections 3 times at one-week intervals. Clones secreting anti-HBcAg were identified by immune adherence hemagglutination (IAHA) and reverse passive hemagglutination (RPHI) techniques. The number of monoclonal antibodies detected by both tests was 18. Furuya et al. do not teach use of a competitive assay for detection of monoclonal antibodies to HBcAg in hybridoma supernatants.
Furuya et al. disclose labeling the monoclonal antibodies with peroxidase and utilizing them in an inhibition assay to determine anti-HBcAg antibodies in serum. Serum containing anti-HBcAg antibodies was incubated with an optimal dose of HBcAg. Glass beads coated with anti-HBcAg monoclonal antibody were added, followed by peroxidase labeled anti-HBcAg monoclonal antibody then o-phenylenediamine. The enzyme reaction was stopped and absorbance (optical density, O.D.) was measured. Percent inhibition was calculated as follows: ##EQU1##
The Furuya et al. assay is a double antibody assay. The immobilized and labeled monoclonal antibodies must bind to different sites on the HBcAg molecule. If they bound to the same site a false positive would result because the binding site would be occupied by the immobilized antibody and the labeled antibody could not bind. This means the immobilized and labeled antibodies must either be different and specific for different epitopes on the HBcAg molecule, or they must be specific for an epitope which occurs more than once on the HBcAg molecule. Perhaps because of these limitations, it is believed that assays for anti-HBcAg of the double monoclonal antibody type have not been commercialized.
In both Tedder et al. and Furuya et al. the anti-HBcAg monoclonal antibodies were produced by immunization of mice with human HBcAg. The purification of HBcAg from human liver or plasma is laborious and constitutes a significant health hazard. Murray et al. (EP 0,013,828 published Aug. 6, 1980) disclose an E. coli transformed with recombinant DNA coding for HBcAg. The polypeptide expressed by this transformed E. coli reacts with human polyclonal anti-HBcAg antibodies and is referred to as rHBcAg.